Pe conjugated anti mouse cd86 antibody

The electrospun nanofiber membranes were first placed in 6-well plates, and RAW264.7 cells were seeded on their surfaces at a density of 2 × 10⁵ cells per well. Following the incubation, the culture medium was discarded, and the cells were washed three times with PBS to remove debris and non-adherent cells. Lipopolysaccharide (LPS, TargetMol) was then added to the culture to activate macrophages. After another 24 h, cells were collected for analysis.
The cell pellets were resuspended in 100 μL of PBS containing 1.25 μg of PE-conjugated anti-mouse CD86 antibody and 0.25 μg of FITC-conjugated anti-mouse CD206 antibody (Biolegend). This step was performed on ice for 30 min to preserve cell viability and prevent nonspecific antibody binding. The expression of CD86 and CD206 was subsequently analyzed using the CytoFLEX flow cytometry system (Beckman Coulter). Additionally, the intracellular ROS level was detected using 2,7dichlorodihydrofluorescein diacetate (DCFH-DA; Yeasen), following the same procedure as described above (n = 3).

RAW264.7 cells were detected by flow cytometry. Fc receptors of the above cells were first blocked with anti-mouse CD16/32 antibody (BioLegend, San Diego, CA, USA), after which the cells were respectively stained with fluorescent antibodies, including PE-conjugated anti-mouse CD86 antibody (BioLegend, San Diego, CA, USA), Brilliant Violet 421-conjugated anti-mouse CD206 (BioLegend, San Diego, CA, USA). The fluorescence antibodies were performed using intracellular staining as previously described [54 (link)]. Meanwhile, cells were respectively stained with isotype-matched control antibodies. Finally, prepared samples were measured and analyzed using a Cyto FLEX flow cytometer (Beckman Coulter, Brea, CA, USA).